Xiaoran Li

THE DIFFERENTIATION OF EMBRYONIC STEM CELLS SEEDED ON ELECTROSPUN NANOFIBERS INTO NEURAL LINEAGES

Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.

Nanofiber Scaffolds with Gradations in Mineral Content for Mimicking the Tendon-to-Bone Insertion Site

We have demonstrated a simple and versatile method for generating a continuously graded, bonelike calcium phosphate coating on a nonwoven mat of electrospun nanofibers. A linear gradient in calcium phosphate content could be achieved across the surface of the nanofiber mat. The gradient had functional consequences with regard to stiffness and biological activity. Specifically, the gradient in mineral content resulted in a gradient in the stiffness of the scaffold and further influenced the activity of mouse preosteoblast MC3T3 cells. This new class of nanofiber-based scaffolds can potentially be employed for repairing the tendon-to-bone insertion site via a tissue engineering approach.

Neurite outgrowth on nanofiber scaffolds with different orders, structures, and surface properties.

Electrospun nanofibers can be readily assembled into various types of scaffolds for applications in neural tissue engineering. The objective of this study is to examine and understand the unique patterns of neurite outgrowth from primary dorsal root ganglia (DRG) cultured on scaffolds of electrospun nanofibers having different orders, structures, and surface properties. We found that the neurites extended radially outward from the DRG main body without specific directionality when cultured on a nonwoven mat of randomly oriented nanofibers. In contrast, the neurites preferentially extended along the long axis of fiber when cultured on a parallel array of aligned nanofibers. When seeded at the border between regions of aligned and random nanofibers, the same DRG simultaneously expressed aligned and random neurite fields in response to the underlying nanofibers. When cultured on a double-layered scaffold where the nanofibers in each layer were aligned along a different direction, the neurites were found to be dependent on the fiber density in both layers. This biaxial pattern clearly demonstrates that neurite outgrowth can be influenced by nanofibers in different layers of a scaffold, rather than the topmost layer only. Taken together, these results will provide valuable information pertaining to the design of nanofiber scaffolds for neuroregenerative applications, as well as the effects of topology on neurite outgrowth, growth cone guidance, and axonal regeneration.

Optimization of preparative conditions for poly-DL-lactide- polyethylene glycol microspheres with entrapped Vibrio cholera antigens.

Poly-dl-lactide-polyethylene glycol (PELA) with different contents of polyethylene glycol(PEG) were synthesized and the PEG content was estimated according to the integral height of hydrogen shown in 1H-NMR. PELA microspheres containing V. cholera antigen, outer membrane protein (OMP) were prepared by a water-in-oil-in-water (W/O/W) based on solvent evaporation procedure. Antigen microspheres with smooth surface, suitable size for oral administration (0.5-5 microm), high loading efficiency (about 60%) and low level of residual solvent (lower than 20ppm) were obtained. Microspheres prepared from PELA with PEG content of about 10% achieved the highest loading efficiency among PELA copolymers and poly-dl-lactide (PLA) homopolymer, which suggested that microspheres size, morphology and the precipitation rate of polymer showed considerable relations with OMP loading efficiency. The regulation of the solvent components of the oil phase contributes to a stable emulsion W/O, and it is concluded that the stable emulsion W/O plays a significant role in improving the protein loading efficiency of obtained microspheres. The addition of stabilizer, such as gelatin and polyvinyl alcohol, into the internal water phase before emulsification produced no significant difference in OMP entrapment and microspheres size. A higher OMP loading efficiency was achieved by adding NaCl or adjusting the pH at the iso-electric point of OMP in the external water phase. It was indicated in vitro that PELA microspheres with smaller size showed larger extent of initial release and higher release rate, whereas microspheres with the diameter of 2.17 microm showed no apparent burst effect.

Nerve Guidance Conduits Based on Double-Layered Scaffolds of Electrospun Nanofibers for Repairing the Peripheral Nervous System

Compared to the nerve guidance conduits (NGCs) constructed from a single layer of aligned nanofibers, bilayer NGCs with random and aligned nanofibers in the outer and inner layers are more robust and tear-resistant during surgical procedures thanks to an isotropic mechanical property provided by the random nanofibers. However, it remains unclear whether the random nanofibers will interfere with the aligned nanofibers to alter the extension pattern of the neurites and impede regeneration. To answer this question, we seeded dorsal root ganglia (DRG) on a double-layered scaffold, with aligned and random nanofibers on the top and bottom layers, respectively, and evaluated the outgrowth of neurites. The random nanofibers in the bottom layer exerted a negative impact on the extension of neurites projecting from the DRG, giving neurites a less ordered structure compared to those cultured on a single layer of aligned nanofibers. The negative impact of the random nanofibers could be effectively mitigated by preseeding the double-layered scaffold with Schwann cells. DRG cultured on top of such a scaffold exhibited a neurite outgrowth pattern similar to that for DRG cultured on a single layer of aligned nanofibers. We further fabricated bilayer NGCs from the double-layered scaffolds and tested their ability to facilitate nerve regeneration in a rat sciatic nerve injury model. Both histomorphometric analysis and functional characterization demonstrated that bilayer NGCs with an inner surface that was preseeded with Schwann cells could reach 54%, 64.2%, and 74.9% of the performance of isografts in terms of nerve fiber number, maximum isometric tetanic force, and mass of the extensor digitorum longus muscle, respectively. It can be concluded that the bilayer NGCs hold great potential in facilitating motor axon regeneration and functional motor recovery.

Promotion of neuronal differentiation of neural progenitor cells by using EGFR antibody functionalized collagen scaffolds for spinal cord injury repair

The main challenge for neural progenitor cell (NPC)-mediated repair of spinal cord injury (SCI) is lack of favorable environment to direct its differentiation towards neurons rather than glial cells. The myelin associated inhibitors have been demonstrated to promote NPC differentiation into glial lineage. Herein, to inhibit the downstream signaling activated by myelin associated inhibitors, cetuximab, an epidermal growth factor receptor (EGFR) neutralizing antibody, functionalized collagen scaffold has been developed as a vehicle for NPC implantation. It was found that collagen-cetuximab 1 μg scaffolds enhanced neuronal differentiation and inhibited astrocytic differentiation of NPCs exposed to myelin proteins significantly in vitro. To test the therapeutic effect in vivo, NPCs expressing green fluorescent protein (GFP)-embedded scaffolds have been implanted into the 4 mm-long hemisection lesion of rats. We found that the collagen-cetuximab 5 μg scaffolds induced neuronal differentiation and decreased astrocytic differentiation of NPCs, enhanced axon regeneration, and promoted functional recovery markedly. A well-functionalized scaffold was constructed to improve the recovery of SCI, which could promote the neuronal differentiation of neural progenitor cells in vivo.

A collagen-binding EGFR single-chain Fv antibody fragment for the targeted cancer therapy.

Collagen, a primary component of the extracellular matrix (ECM), is highly expressed in a variety of cancers and influences the tumor microenvironment by increasing the recruitment of macrophages and endothelial cells. Therefore, collagen is a highly promising target for cancer therapy. The collagen-binding domain (CBD) can dynamically bind to collagen and achieve the sustained release of CBD-fused protein in the collagen network. Here, we developed a collagen-binding epidermal growth factor receptor (EGFR) antibody fragment for targeting the collagen-rich ECM in tumors. The single chain fragment variable (scFv) of cetuximab was fused to CBD (CBD-scFv) and expressed in Pichia pastoris. CBD-scFv preserved the antigen binding domain and anti-tumor activity of cetuximab in vitro. Moreover, CBD-scFv displayed a collagen binding ability due to the function of CBD. In vivo experiments revealed that CBD-scFv bound to collagen and achieved sustained release in tumors. Furthermore, CBD-scFv significantly suppressed the growth of tumors in A431 xenografts. Therefore, CBD-scFv had a potential therapeutic value for the collagen-rich carcinomas. The specific target and sustained release of CBD-scFv in tumors could be a new approach for targeted drug delivery in cancer therapy.

The miR-7 Identified from Collagen Biomaterial-Based Three-Dimensional Cultured Cells Regulates Neural Stem Cell Differentiation

Increasing evidence suggests that three-dimensional (3D) cultures provide more appropriate microenvironments to control stem cell response compared with traditional two-dimensional (2D) cultures. However, the molecular mechanism involved in 3D cultured stem cells is not well known. Several microRNAs whose target genes involved in the regulation of self-renewal and differentiation of stem cells were found to be downregulated in 3D cultured PA-1 cells. Among them, miR-7 was predicted to target Kruppel-like factor 4 (Klf4), a key gene for self-renewal of neural stem cells (NSCs). We showed that the differentiation of NSCs was inhibited in 3D collagen scaffolds compared with 2D cultured cells. The quantitative real-time PCR (qPCR) analysis indicated that the expression of miR-7 and Klf4 changed significantly in 2D cultures, whereas the expression stability of miR-7 and Klf4 was detected in 3D cultures. Using luciferase assay and western blot, Klf4 was identified as a target of miR-7 indicating that miR-7 plays a critical role in maintaining the self-renewal capacity through a Klf4-dependent mechanism in 3D cultured cells. Thus, the collagen scaffold-based 3D cell cultures may provide a platform to reveal the regulatory mechanism of cell regulators, which are difficult to find in traditional 2D cell cultures.

Electrospun Collagen Fibers with Spatial Patterning of SDF1α for the Guidance of Neural Stem Cells

Producing gradients of biological cues into nerve conduits is crucial for nerve guidance and regeneration. Herein, the fabrication of gradients of stromal cell-derived factor-1α (SDF1α) on electrospun collagen mats is reported using an electrohydrodynamic jet printing technique. The fabrication of various SDF1α gradated patterns on collagen fibrous mats is successfully demonstrated including shallow continuous gradient, steep continuous gradient, and step gradient by controlling the processing parameters. The SDF1α graded collagen scaffolds show a long-term stable gradient, as SDF1α is fused with a unique peptide of collagen binding domain (CBD), and CBD-SDF1α can specifically bind to the collagen mat. Such graded scaffolds exhibit sustained release of SDF1α. Further examination of neural stem cell (NSC) response to the CBD-SDF1α gradients with various patterns show that the NSCs can sense the CBD-SDF1α gradients, display a polarized morphology, and tend to migrate toward the region with a higher CBD-SDF1α content. The collagen mats with CBD-SDF1α gradients guide gradual distribution of NSCs, and NSC-differentiated neurons and astrocytes after seeding for 1 and 7 d. This new class of CBD-SDF1α gradient scaffolds can potentially be employed for guided nerve regeneration.

Maintenance of the self-renewal properties of neural progenitor cells cultured in three-dimensional collagen scaffolds by the REDD1-mTOR signal pathway

Three-dimensional (3-D) culture, compared with traditional two-dimensional (2-D) cell culture, can provide physical signals and 3-D matrix close to the in vivo microenvironments. Here, sponge-like collagen scaffolds were used to assess how 3-D culture would affect the differentiation and self-renewal of neural progenitor cells (NPCs). Cultured in differentiation medium without growth factors, cells in 3-D collagen scaffolds yielded much higher clone formation efficiency and expressed less neuron marker, TUJ1, compared with cells cultured on 2-D plates. mTOR inactivation was identified and showed to supported the self-renewal of NPCs in 3-D culture. At the same time, REDD1 was highly expressed in cells cultured in 3-D conditions, which blocks the activity of mTOR. Moreover, knocking-down REDD1 induced the differentiation of NPCs in 3-D collagen scaffolds. These results indicated that mTOR inactivation by REDD1 mediated the self-renewal regulation of NPCs in 3-D cultures. Thus, 3-D collagen scaffolds maintained self-renewal properties of NPCs, and the inhibitory regulator of mTOR (such as REDD1) played an important role in the regulation of self-renewal and differentiation of NPCs.